1. Field of the Invention
The invention pertains to the field of erectile dysfunction. More particularly it relates to cells useful for restoring erectile function including stem cells, regenerative cells, and angiogenesis promoting cells. More specifically, the invention relates to cord blood, bone marrow, peripheral blood, and specialized progenitor cells and products thereof.
2. Description of the Related Art
Erectile Dysfunction
Erectile dysfunction (ED) is characterized by the lack of ability to achieve and maintain penile erection for intercourse. The prevalence of ED is strongly correlated with increasing age. For example, according to the Massachusetts Male Aging Study, 67% of men 70 years of age experience some form of ED, whereas at age 40 only 39% of men report this [1]. ED is typically scored based on symptomology, although quantitative measurement instruments exist such as the Erectile Function Visual Analog Scale (EF-VAS) and the International Index of Erectile Function [2, 3]. An estimate of the number of Americans suffering from ED is believed to range from 10-30 million [4]. The prevalence of ED is illustrated by the fact that over 39 million prescriptions have been written for one ED drug, Viagra, in the United States as of 2006. World-wide it is believed that 100 million men are affected by various degrees of ED. Currently ED is treated by oral inhibitors of phosphodiesterase V, which is considered the standard of care. Despite this, numerous patients are unresponsive to treatment, do not tolerate adverse effects associated with treatment, or are ineligible for treatment. In order to properly overview the prior art relating to treatment of ED, and to provide a basis for the practitioner of the invention to utilize the invention disclosed optimally, it is necessary first to review the basic biology of the penis as relating to erections.
Penis Anatomy and Erection
The penis is comprised of three erectile bodies, 2 parallel ones termed corpora cavernosa, and underneath, wedged in between, the corpus spongiosum, which contains the urethra. The three erectile bodies are heavily vascularized and contain large proportion of smooth muscle cells. Erection is caused by neurologically-induced relaxation of smooth muscle cells in the erectile bodies, which allows influx and accumulation of blood into the balloon-like sacs between the smooth muscle cells called sinusoids. As blood accumulates, the outflow of blood is prevented by pressure from the tunica albuginea against the venous plexus, thus causing trapping of the blood, allowing erection to occur. The process of blood accumulation due to venous trapping is termed the veno-occlusive mechanism. Additional rigidity of the penis shaft is provided by contraction of the ischiocaverous muscles.
The initial nerve impulses triggering the erection originate from the brain in response to sexual stimuli. Although currently this area is under investigation, certain parts of the brain have been identified responsible for arousal. These include structures such as the frontal lobe, the hypothalamus, thalamus, amygdala and the cingulated gyrus [5]. The first identification of a brain structure associated with erection occurred in the 1960s in primate experiments using brain electrostimulation via a stereotactic technique. These studies demonstrated that stimulation of the medial frontal lobe led to erections in a consistently reproducible manner [6]. Subsequent clinical experiments demonstrated that during exposure to sexual stimuli, functional MRI could accurately identify increased perfusion activity in the frontal lobe, the cingulated gyrus, and the thalamus [7]. Interventional evidence of the importance of distinct anatomical areas in the central control of erection comes from clinical studies showing deep brain stimulation of the thalamic area for treatment of Tourette's syndrome leads to unexpected erections as a consequence [8]. Furthermore, the successful use of centrally acting drugs such as apomorphine in the treatment of ED, supports the importance of cerebral control of erection [9]. Older studies in pedophiles and rapists reported some success at the central inhibition of abnormal sex drive through the surgical ablation of certain anatomical regions of the hypothalamus [10].
In contrast to central regulation of erections, much more is known about spinal control. It is established that the sacral parasympathetic nucleus, the thoracolumbar sympathetic nuclei and the pudendal motoneurons are all involved the stepwise transmission of signals between the dorsal penile nerves and central nervous system. Identification of specific areas was performed in studies, such as Stief et al, who showed that electrostimulation of the roots of S2 to S55 in man with complete thoracic spinal injury, was able to produce erection in all patients assessed [11]. A variety of electro-spinal stimulation of erection studies were reviewed by Giuliano et al [12]. From these studies it was identified that during normal erection, parasympathetic nervous activation causes relaxation of smooth muscle and dilation of the helicine arteries in the corpora cavernosum and the corpus spongiosum. This dilation, in combination with the veno-occlusive mechanism which prevents blood outflow from the penile body causes the end result of erection. Mechanistically, parasympathetic activation causes upregulation of nitric oxide (NO) production by nonadrenergic, noncholinergic nerves, as well as endothelium which lines the penile arteries and cavernosal sinusoids. Accumulation of NO increases production of cyclic guanosine monophosphate (cGMP) through activation of the enzyme guanylyl cyclase. cGMP acts as a second messenger which leads to decrease calcium uptake into the cavernous and endothelial-lining smooth muscles, thus causing relaxation and hence erection [13]. Since phosphodiesterase (PDE)-5 is involved in the breakdown of cGMP, the inhibition of PDE-5 has been chosen as a pharmacological goal of medications such as Viagra (sildenafil), Cialis (tadalafil) and Cialis (vardenafil) [14].
Causes of Erectile Dysfunction
It is known that ED is multifactorial, with causative influences including vasculogenic, endocrinological, psychogenic, and neurogenic. Some reports cite vascular disease as being culprit in as many as 85% of cases either directly or indirectly [15]. Accordingly this discussion begins with the endothelial cell dysfunction associated with ED. Vascular disease is associated with either decreased production of NO, or decreased responsiveness to its actions. There are 5 mechanisms postulated for decrease in this intermediate, all 5 associated with ED:
The first is oxidative stress in the form of the oxygen free radical superoxide, which both enhances degradation of NO (by direct conversion to peroxynitrite), as well as decreases its synthesis [16]. Importance of superoxide in induction of erectile dysfunction was demonstrated in experiments where administration of exogenous superoxide dismutase was able to increase erectile function in a model of rat diabetes associated ED [17].
The second mechanism is preformed advanced glycation end products, which are found in diabetics, as well as at higher concentrations in elderly patients [18]. These inactivate NO directly [19], induce an increased production of superoxide [20], which also inhibit NO as previously mentioned, and directly suppress synthesis of endothelial nitric oxide synthase (eNOS) by endothelial cells [21].
The third mechanism is enhanced expression of the enzyme arginase II [22], which compete with nitric oxide synthase for arginine. Thus enhanced arginase expression, which is associated with ED leads to inhibition of NO.
The forth mechanism is reduced transcription of eNOS and nNOS in tissue lacking testosterone [23, 24].
The fifth mechanism is increased activity of the Rho/Rho kinase which is associated with atherosclerosis. The Rho/Rho kinase both inhibits NOS activity and increases vascular smooth muscle tone, thereby inhibiting NO and erectile ability, respectively [25].
In all of the above five mechanisms, endothelial dysfunction seems to be the primary cause. Endothelial dysfunction is induced by aging, artherosclerotic changes, and oxidative stress. The essential role of the endothelium in ED is supported by studies, which demonstrate that ED onset is a precursor to other more serious cardiovascular diseases such as coronary heart disease before disease symptoms arise. For example in a study of 221 patients who were referred to undergo stress myocardial perfusion single-photon emission computed tomography (MPS), patients were screened for ED using a validated questionnaire. 54.8% of the screened patients demonstrated advanced ED. Patients with ED presented a higher level of coronary heart disease in comparison to patients without ED in terms of a summed MPS score>8, (43.0% vs 17.0%, respectively). ED patients also bad LVEF lower than 50% (24.0% vs 11.0%), shorter exercise time (8.0 vs 10.1 minutes) and lower Duke treadmill score (4.4 vs 8.4; P<0.001) in comparison to patients without ED. The role of ED as an independent predictor of severe coronary heart disease was identified using multivariate analysis (odds ratio, 2.50; 95% confidence interval, 1.24-5.04; P=0.01) [26]. In another study, 12 men with ED (IIEF-5 questionnaire score</=18) and 12 age-matched controls (IIEF-5 questionnaire score>/=21) were assessed for coronary flow velocity reserve by Doppler in the left anterior descending artery, before and during adenosine infusion. Flow velocity reserve was significantly reduced in subjects with erectile dysfunction: 2.36 versus 3.19; P=0.024. Using multivariate analysis, adjusting for age, tobacco use, systolic blood pressure, heart rate and body mass index, ED was the only significant predictor of reduced coronary flow velocity reserve, P=0.016 [27]. In another study, the severity of ED was compared with risk of heart disease. Men were stratified for risk for heart disease within 10 years using the Framingham risk profile algorithms. In the heart disease risk cohort men with moderate/severe ED (IIEF5 5-16) had a 65% increased relative risk for developing CHD within 10 yrs compared to those without ED (IIEF5 22-25). These data prompted the authors to state “Moderate to severe ED, but not mild ED is associated with a considerably increased risk for coronary heart disease” [28]. The possibility of ED being a predictive factor for development of more serious vascular diseases has prompted Montorsi et al to put forth the “Artery Size Hypothesis”, in which the authors propose that smaller arteries, such as the pudendal arteries supplying blood to the penile structures, are more likely to be effected by artheroscierotic and other forms of endothelial damage, in comparison to larger vessels. Accordingly, the initiation of vascular disease is first identified in many cases as ED, which subsequently progresses to more advanced diseases. The authors make 4 points supporting this hypothesis, namely: a) ED and coronary artery disease should be considered as two different manifestations of the same disease process; b) Prevalence of occult coronary artery disease in ED should be low; c) Prevalence of ED in patients with coronary artery disease should be high; and d) Coronary artery disease should occur subsequently to ED in a wide variety of patients. The authors cite numerous supportive studies in a publications explaining their hypothesis [29].
The importance of the endothelial dysfunction in ED is exemplified by numerous assays, which have demonstrated not only correlation between dysfunction and ED, but also between reversion of ED and increased endothelial function. Studies demonstrating endothelial dysfunction include observations of reduced brachial flow dilation in ED patients [30, 31], reduced reactive hyperemic response [32], impaired mean blood pressure and platelet aggregation responses to L-arginine [33] and reduced endothelial precursor cells in circulation [34, 35]. Interventions that successful treat some forms of ED such as PDE5 inhibitors have been shown to increase both the numbers of circulating endothelial progenitors cells [36], as well as the brachial flow mediated dilation response [37-40]. Interestingly exercise [41], administration of statin drugs [42, 43], as well as pregnancy [44], has been demonstrated to correlate with increased numbers of circulating endothelial cells.
In animal models, administration of agents capable of inducing endothelial cell proliferation, and/or neoangiogenesis, induces inhibition of ED progression or reversion of ED. For example, basic fibroblast growth factor (bFGF) is a known inducer of angiogenesis in ischemic situations, and its exogenous administration is therapeutic in models of stroke [45], angina [46], and peripheral limb ischemia [47]. The administration of two 2.5 microgram doses of bFGF, separated by a 3-week interval into corporal tissue of hypercholesterolemic rabbits was shown to increase corporal relaxation in response to chemical stimuli, as well as ability to generate NO [48]. In another study, administration of bFGF intracavernously into diabetic rats by means of gelatin microbeads resulted in protection of erectile function from diabetes mediated onset [49]. Yet another study demonstrated that systemic basic fibroblast growth factor induces favorable histological changes in the corpus cavernosum of hypercholesterolemic rabbits [50].
Current Treatments of ED
The current standard of care for treatment of ED is phosphodiesterase inhibitors. For example, U.S. Pat. No. 5,250,534 discloses sildenafil (VIAGRA), an orally available PDE5 inhibitor. Additional PDE5 inhibitors include, Cialis (tadalafil) and Cialis (vardenafil). Unfortunately, a substantial number of patients (20-40%) are resistant to PDE5 inhibitors. These include substantial numbers of patients with advanced neurologic damage, diabetes mellitus, or vascular disease. This may be due to the fact that inhibition of PDE5 upregulates erectile mechanisms such as potentiating the effects of NO, but still depend on functional erectile tissue to be present in substantial concentrations. For example, a high adipose to smooth muscle ratio in cavernous tissue, as well as decreased expression or activity of neuronal or endothelial NO synthase (NOS), impaired NO release, accelerated NO destruction, and atrophy of cavernosal structures is associated with resistance of ED patients to PDE5 inhibitors [51, 52]. Accordingly, there is a need to develop means of regenerating erectile tissue, or components thereof in a natural and physiological manner as a means of treating ED instead of augmenting the activity of the already diminished tissue existing in patients with ED.
PDE5 inhibitors are known to possess a variety of systemic effects in numerous organ systems, therefore the long term effects of PDE5 inhibition are still uncertain. It is known that PDE5 inhibitors can induce a variety of adverse effects such as optic neuropathy [53], headaches [54], and various cardiovascular pathologies [55], especially when taken in combination with nitrates [56]. In fact, in 1998, the US Food and Drug Administration published a report on 130 confirmed deaths among men who received prescriptions for sildenafil citrate, with causes of death included arrythmias, sudden cardiac death and hypotension-associated events [57]. Beneficial non-ED uses of PDE5 inhibitors are known, for example, since PDE5 is expressed in lung tissue, investigators sought to, and succeeded at inhibiting symptomatic pulmonary arterial hypertension in a double blind clinical trial [58] by administration of sildenafil citrate. However, given the various areas in the body that PDE5 is expressed, such as platelets, kidneys, and pancreas [59], it is the belief of some that systemic inhibition of this enzymatic system may have physiologic consequences in the long-run [60].
Numerous other therapeutic products have been applied previously and some are still in use for treatment of ED. Non-pharmacological methods include the use of vacuum pumps, as well as penile prosthetic surgery. A penile prosthesis is described in U.S. Pat. No. 5,065,744.
Pharmacological methods include intracavernous administration of CAVERJECT (U.S. Pat. No. 4,127,118) and intra-urethral administration of MUSE (U.S. Pat. No. 5,773,020) in which the active ingredient is PGE-1. Two serious adverse reactions associated with these agents are drug-induced pain and priapism. Specifically it is known that single use of PGE1 therapy produces a pain response in about 3 to 10 percent of patients, which increases with continued use. Additionally, manipulation of the penis prior to intercourse is not desirable by many patients. Treatments using intracavernosal injections are associated with a variety of adverse effects, which with increased occurrence have profound consequences. Said adverse effects include cutaneous ecchymosis, corporal fibrosis, and in some situations damage to the penile nerves. Pharmacological treatment methods that have been patented but have not been clinically approved include: a) U.S. Pat. No. 3,943,246, which describes treatment of impotence administration of oxytocin; U.S. Pat. No. 4,530,920 which describes nonapeptide and decapeptide analogs of luteinizing hormone releasing hormone agonists for treatment of ED; c) U.S. Pat. No. 4,139,617 which describes 19-oxygenated-androst-5-enes treatment of ED; and d) U.S. Pat. No. 5,541,211 which describes the use of yohimbine in treatment of ED.
Devices for intrascrotal implantation have been described in U.S. Pat. No. 5,518,499, which allow for administration of vasoactive agents such as PGE-1 without the need for intracavernousal injections. However this approach is highly invasive and does not treat the physiological cause of ED.
Centrally acting treatments for erectile dysfunction include apomorphine, as disclosed in U.S. Pat. No. 5,770,606. U.S. Pat. No. 4,801,587 discloses that phentolamine (VASOMAX®) which is available in a number of countries for treating hypertension is also useful for treating ED.
Details Regarding Diagnosis and Assessment of ED
This section will describe some of the details associated with diagnosis of ED. Although the invention describes numerous means of treatment of ED, it is important for the practitioner of the invention to realize the broad spectrum of diagnostic tools so that they will be able to decide which fit their respective patient populations.
ED is usually first assessed by the physical examination with special attention to possible penile and scrotal pathology or abnormalities, which may alter or inhibit the ability of the penis to cause penetration. Anatomical abnormalities or visible injuries are usually a small percentage of causative factors in ED. Subsequently, more detailed tests are performed such as the penobrachial blood pressure index (PBPI), doppler investigation of the penile arteries, and the papaverine test. These are described in detail in U.S. Pat. No. 6,132,757 and incorporated by reference herein. The PBPI is the penile systolic blood pressure divided by the systolic blood pressure determined at one of the arms. These blood pressures can be determined by any number of standard techniques. Thus, the penile systolic blood pressure can be determined by i) placing an inflatable cuff around the base of the free part of the penis in the flaccid state which is capable of being used to apply variable pressure, readable from a gauge, to an object around which the cuff is placed, ii) localizing the penile arteries with a Doppler ultrasound probe (e.g., 8 MHz probe, such as the Mini Doplex D500 available from Huntleigh Technology, Luton, United Kingdom), and then iii) inflating and deflating the cuff and ascertaining the pressure at which the Doppler sound reappears. The pressure at which the Doppler sound reappears is the penile systolic blood pressure. A male's penile blood pressure is regarded as normal if his PBPI is >0.80. With regard to Doppler investigation, each of the two penile cavernous arteries is investigated distal to the aforementioned cuff using the Doppler ultrasound problem. The function of each of the two arteries is assessed by Doppler ultrasound using an arbitrary scale of 0, 1, 2 or 3, where 0 means that the function is so deficient that the artery cannot be located and 3 means that the artery is well enough that maximal Doppler sound is observed. Detailed description of Doppler analysis and the papaverine tests are given in the following references [61-64]. The use of the Erectile Function Visual Analog Scale (EF-VAS) has become the golden standard for diagnosis and evaluation of treatment efficacy since it is based on overall functionality and not specific anatomical or biological abnormalities/defects.